Integrated Microfluidic Chips For Single-cell Gene Expression Profiling

Micro and Nano science and technologies refer to a collection of functionalized structures, devices or systems, and the investigation of the corresponding issues, which defines the manufacturing frontier of a nation. Cells are the fundamental units of biological processes. Gene expression profiling at the single-cell level, which is critical to understanding the complex pathogenesis and evolution of intractable diseases, and advancing drug discovery or development, is an ultimate analytical technology in life science. Most recently, single-cell analysis has been burgeoned as a cutting edge in fundamental biology and clinical settings. In this paper, we combined MEMS(Micro-Electro-Mechanical System) fabrication technology with the single-cell research by fully integrating all steps of biochemical assays in a microchip. Isolation, purification, amplification and analysis of low-abundance m RNA from individual cells have been performed. The significance, novelty and main content of this paper are shown as follows.Two microfluidic systems with improved simplicity and versatility have been designed(i.e. single-unit chips and arrayed microfluidic chips). The former is more portable and easy-to-use while the latter increases testing throughput and operating efficiency. Multilayer architectures including flow layer, control layer, interlayer and microheater/sensor have been employed by these systems. Also, the fabrication of these chips have been improved to restrict common issues in existing methods such as PDMS thin film adhesion, reagent evaporation during thermal cycling and the reusability of heater/sensors. Furthermore, based on the heat transfer theory, the designing performance of the heater/sensor has been evaluated.Then, a customer tailored experimental system including microfluidic control, real-time temperature control, biological samples preparation and real-time fluorescent molecule detecting platforms has been set up. Of these, the microfluidic control platform contains syringe pumps, liquid Nitrogen tank and a microscope with white light source. The temperature control platform was based on the virtual instrument technology and employed Lab VIEW programming to construct customer control interfaces and panels that is able to detect and modulate on-chip temperature in real time. Biological sample preparation platform combines cell incubator, laminar bio-hood and cell centrifuge to assist cell culture and passage, drug treatment and reagents mixing. Fluorescent molecule detecting platform includes a motorized inverted microscope, CCD and the software for image capturing and analysis, with properties of high single-to-noise ratio(SNR), applicability and sensitivity.In parallel, as the hydrodynamic effect on live cells which affects cell viability significantly is difficult to quantify experimentally, the thesis incorporated the theoretical models of Computational Fluid Dynamics, Elasticity and Plasticity in 2 and 3-dimentsion, and employed Arbitrary Lagrangian-Eulerian description, moving mesh method and parametric solver to study the Fluid-Structure interaction of cell and carrier flow in the microsystems. The transient cell isolation and immobilization process have been simulated, and then the hydrodynamic forces distributed on cell surface were quantified. Also, the correlations of inlet flow rate with these hydrodynamic forces were plotted out. Furthermore, the simulation results were compared with the extracellular pressure in human vascular and interstitial fluid to evaluate cell viability impairment in the microsystems.In addition, a fully integrated approach for single-cell gene expression has been presented in this thesis. In the microchips, released m RNA templates from individual cells of human breast cancer cell line MCF-7 was isolated, purified, amplified and analyzed. Steps of cell capture and lysis, solid-phase based m RNA isolation, c DNA synthesis and amplification were all integrated which reduced operating complexity while increased the fidelity of low-abundance m RNA. Therefore, the approach improved testing accuracy, sensitivity and SNR. In experiments, we assessed the feasibility and intra-assay reproducibility of the approach followed by optimization of cell trapping and lysis procedures and compared the efficiency, sensitivity and yield of the approach with traditional methods subsequently. Furthermore, the variation in gene expression levels of housekeeping gene GAPDH and cell cycle regulated gene CDKN1 A after MMS treatment has been measured by the presented approach. Also, the pharmacokinetics and gene expression regulation by the alkylating agent has been investigated. Finally, the effects of drug dosage and treating time length on the expression levels of CDKN1 A gene from single MCF-7 cells have been quantified and analyzed.The presented microchips and approach improved the integrity, versatility and applicability of the existing reports. Meanwhile, the device design, fabrication and operation have been simplified significantly, and therefore the complexity, costs and reagents consumption have been minimized. The study is laboratory oriented at this stage and can be commercialized in the future. Thus, the presented devices and approach is capable of promoting the combination of microfluidics technology with fundamental study, and hold a potential to improve biomedical research and release mysterious issues in human health.